The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy
Eugenio Montini, Daniela Cesana, Manfred Schmidt, Francesca Sanvito, Cynthia C. Bartholomae, Marco Ranzani, Fabrizio Benedicenti, Lucia Sergi Sergi, Alessandro Ambrosi, Maurilio Ponzoni, Claudio Doglioni, Clelia Di Serio, Christof von Kalle, Luigi Naldini
Eugenio Montini, Daniela Cesana, Manfred Schmidt, Francesca Sanvito, Cynthia C. Bartholomae, Marco Ranzani, Fabrizio Benedicenti, Lucia Sergi Sergi, Alessandro Ambrosi, Maurilio Ponzoni, Claudio Doglioni, Clelia Di Serio, Christof von Kalle, Luigi Naldini
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Research Article

The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy

Abstract

γ-Retroviral vectors (γRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between γRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of γRVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as γRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by γRVs. This integration-site bias was intrinsic to γRVs, as it was also observed for SIN γRVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.

Authors

Eugenio Montini, Daniela Cesana, Manfred Schmidt, Francesca Sanvito, Cynthia C. Bartholomae, Marco Ranzani, Fabrizio Benedicenti, Lucia Sergi Sergi, Alessandro Ambrosi, Maurilio Ponzoni, Claudio Doglioni, Clelia Di Serio, Christof von Kalle, Luigi Naldini

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Figure 2

Survival curves related to vector treatment.

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Survival curves related to vector treatment. (A and B) Kaplan-Meier surv...
(A and B) Kaplan-Meier survival curves of mice transplanted with cells transduced with SF.LTR vectors (A) and SIN.LTR vectors (B). For each panel, the survival curve of the mock group is shown (n = number of transplanted mice). Survival of the LV.SF.LTR, MOI = 100, group was significantly shorter than that of the mock group (P < 0.0001; Mantel-Cox log-rank test). (C) Survival probability over time was calculated for each group using the estimated log-logistic parameter (shown in Table 2) and compared with that of the mock group. The survival probability of the LV.SF.LTR, MOI = 100, and the RV.SF.LTR groups was significantly lower than that of the mock group (P values are indicated). A sample of the transduced cells was kept in vitro for 2 weeks after transduction to measure the average VCN (shown for reference).